The aim of this project is to obtain a mechanistic understanding of the roles of the enzyme, iron and s-adenosylmethionine (SAM) in the reaction catalyzed by the pyruvate formate-lyase activating enzyme (AE). The pyruvate formate-lyase activating enzyme utilizes iron and SAM to generate a stable glycyl radical on pyruvate formate-lyase (PFL), thereby activating it for normal biological function. While kozarich and coworkers have demonstrated stoichiometric binding of Fe(II) to AE, little is known about the metal coordination environment or the role of the iron in radical generation. As described in the proposal, AE appears to be one of an emerging class of enzymes, all of which utilize iron and SAM to generate radicals or catalyze radical-mediated reactions. In many cases these reactions are analogous to those catalyzed by adenosylcobalamin- dependent enzymes, and thus questions have arisen regarding the potential mechanistic similarities between adenosylcobalamin and iron/SAM-mediated enzymatic reactions. preliminary studies described in this proposal demonstrate that AE, like the other members of ht iron/SAM class of enzymes, contains an iron-sulfur cluster. The work described in this proposal has as its specific goal the characterization of the AE metal center and the elucidation of the role of the metal center and SAM in generating a radical on PFL. in a more general sense these studies will also lead to the development of a mechanistic understanding of this new class of iron and SAM-dependent enzymes. The specific aims of the proposal are as follows: 1. To determine the requirements for maximal specific activity by investigating the expression system and the expression conditions. 2. To characterize the iron-sulfur cluster in pyruvate formate-lyase activating enzyme (AE). The approach will include examination of native AE with UV-Vis, MCD, EPR, Raman, and Mossbauer spectroscopy. 3. To examine the role of Fe(II) in cluster conversion and/or in AE catalytic activity. The question of the role of the added Fe(II) in the enzyme assay will be addressed using both spectroscopic and kinetic approaches. 4. To probe the biological relevance of the iron-sulfur center of AE using kinetic approaches including: 1. the correlation of enzymatic activity with the iron and sulfide content, and 2, a detailed examination of the requirements for enzymatic activity when an intact iron-sulfur center is present. 5. To utilize the metal chromophore as a spectroscopic probe for the interaction of AE with SAM and PFL. The iron-sulfur center of AE will provide a convenient spectroscopic handle for monitoring alterations in the metal coordination environment upon substrate binding.